Publication 2

Meier J, Nolte G, Schneider TR, Engel AK, Leicht G, et al. (2019) Intrinsic 40Hz-phase asymmetries predict tACS effects during conscious auditory perception. PLoS ONE 14(4): e0213996. https://doi.org/10.1371/journal.pone.0213996

RESEARCH ARTICLE

Intrinsic 40Hz-phase asymmetries predict tACS effects during conscious auditory perception

Jan MeierID¹*, Guido Nolte², Till R. Schneider², Andreas K. Engel², Gregor LeichtID¹, Christoph Mulert^1,3

1 Department of Psychiatry and Psychotherapy, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 2 Department of Neurophysiology and Pathophysiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, 3 Centre for Psychiatry and Psychotherapy, Justus-Liebig-University Giessen, Giessen, Germany

*j.meier@uke.de

Abstract

Synchronized oscillatory gamma-band activity (30-100Hz) has been suggested to constitute a key mechanism to dynamically orchestrate sensory information integration across multiple spatio-temporal scales. We here tested whether interhemispheric functional connectivity and ensuing auditory perception can selectively be modulated by high-density transcranial alternating current stimulation (HD-tACS). For this purpose, we applied multi-site HD-tACS at 40Hz bilaterally with a phase lag of 180˚ and recorded a 64-channel EEG to study the oscillatory phase dynamics at the source-space level during a dichotic listening (DL) task in twenty-six healthy participants. In this study, we revealed an oscillatory phase signature at 40Hz which reflects different temporal profiles of the phase asymmetries during left and right ear percept. Here we report that 180˚-tACS did not affect the right ear advantage during DL at group level. However, a follow-up analysis revealed that the intrinsic phase asymme-tries during sham-tACS determined the directionality of the behavioral modulations: While a shift to left ear percept was associated with augmented interhemispheric asymmetry (closer to 180˚), a shift to right ear processing was elicited in subjects with lower asymmetry (closer to 0˚). Crucially, the modulation of the interhemispheric network dynamics depended on the deviation of the tACS-induced phase-lag from the intrinsic phase asymmetry. Our character-ization of the oscillatory network trends is giving rise to the importance of phase-specific gamma-band coupling during ambiguous auditory perception, and emphasizes the neces-sity to address the inter-individual variability of phase asymmetries in future studies by tai-lored stimulation protocols.

Introduction

Synchronized neuronal activity across widely distributed cortical regions is encoded in unique spectral signatures and thought to reflect a key mechanism for cortical information integration and conscious perception in humans [1]. In particular, synchronization in the gamma-a1111111111

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Citation: Meier J, Nolte G, Schneider TR, Engel AK, Leicht G, Mulert C (2019) Intrinsic 40Hz-phase asymmetries predict tACS effects during conscious auditory perception. PLoS ONE 14(4): e0213996.

https://doi.org/10.1371/journal.pone.0213996 Editor: Ilona Papousek, University of Graz, AUSTRIA

Received: September 26, 2018 Accepted: March 5, 2019 Published: April 3, 2019

Copyright:©2019 Meier et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files.

Funding: This work was supported by grants from the German Research Foundation (SFB936/C6 to G.L., C.M.; SFB936/A3/Z1 to A.K.E.; SPP1665 EN 533/13-1 to A.K.E.; SFB936/Z3 to G.N.; SPP1665/2 SCHN 1511/1-2 to T.R.S.) and the Werner Otto Foundation (J.M.). German Research Foundation:

www.dfg.de. Werner Otto Foundation:http://www.

werner-otto-stiftung.de/de/. The funders had no role in study design, data collection and analysis,

frequency range (30–100 Hz) has been associated with feature integration from distant cortical sites [2] and might efficiently route cortical information flow to task-relevant cortical regions [3]. While most of the previous work was done in the visual domain [1–3], recent findings indicated that a similar mechanism might underlie conscious auditory perception [4,5], where information from both ears is integrated across both auditory cortices during a dichotic listen-ing (DL) task (Fig 1A) [6]. In this paradigm, healthy participants typically exhibit the well-known right ear advantage during DL; they report more often the syllable presented to the right than to the left ear [7], which is best explained by the supremacy of contralateral path-ways from the speech-dominant left hemisphere [8]. Furthermore, left ear percept is associated with increased functional [4] and effective [5] gamma-band connectivity, which might be mediated by cortico-cortical callosal fibers [6].

Even though most of this evidence is correlative in nature, causal links between oscillatory key signatures during auditory processing and structural connections could be investigated with novel non-invasive brain stimulation techniques such as transcranial alternating current stimulation (tACS), which enable frequency-specific modulation of cortical oscillations [9]. In the past, tACS has been suggested to entrain cortical oscillations in a frequency-specific man-ner [10–15] and phase-dependent effects have been demonstrated in human [16–22] and ani-mal studies [10,12], making it an ideal tool to probe the causal influence of phase relationships on conscious auditory perception [23,24]. Importantly, highly selective stimulation at different cortical sites can now be implemented by optimized stimulation protocols derived from computational models [14,18,19].

In this study, we tested whether the interhemispheric information flow during a dichotic listening (Fig 1A) can be modulated by spatially-matched multi-site 40Hz with a phase-lag of 180˚ between the left and right auditory cortex (BA42). Since it has been shown that the inter-hemispheric integration of alternating visual tokens into coherent motion percept can reliably be inhibited by 40Hz-tACS with a phase-lag of 180˚ between hemispheres [18,25], it is con-ceivable that interhemispheric auditory processing could be selectively altered using a similar stimulation protocol with a tailored high density (HD)-electrode montage derived from cur-rent flow modeling (Fig 2). We thus hypothesized that 40Hz-tACS with a phase-lag of 180˚

between hemispheres should inhibit network synchrony and thereby increase the laterality index.

Whilst previous studies support the concept that inter-areal gamma-band synchronization entails a delayed non-zero phase relationship [4,5,26], the associated metrics (lagged phase synchronization [27], isolated effective coherence [28]) however do not permit the deduction of a specific phase asymmetry between the left and right auditory cortices in degree notation.

To address this issue, we employed an exploratory control analysis to establish a link between the behavioral outcome of the anti-phase stimulation and the individual phase asym-metry during the sham session, recorded with 64-channel electroencephalography (EEG).

Hence we investigated whether the time courses of the intrinsic phase asymmetry at 40Hz dif-fered between left and right ear percept, and specifically assessed the circular-linear correlation between the intrinsic phase asymmetry and the behavioral tACS-related modulation.

Materials and methods Participants

Twenty-nine healthy participants were recruited from the University Medical Center in Ham-burg, Germany. All subjects were right-handed according to the Edinburgh handedness-scale [29], reported no history of neurological or psychiatric disease, filled out a sociodemographic questionnaire and further provided written informed consent and were paid for participation.

Intrinsic phase asymmetries predict tACS effects

decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Please note that we assessed sociodemographic data as a standard procedure to allow for potential comparisons of healthy control samples with clinical samples. Since no association between sociodemographic factors and early auditory perception in healthy participants had previously been found, we did not further report these data in this manuscript.

Normal hearing was validated by pure tone audiometry for frequencies between 125 and 8000Hz (Esser Home Audiometer 2.0). No participant exhibited interaural differences larger than 9 dB or an auditory threshold above 25 dB. The study was approved by the ethical com-mittee of Medical Association Hamburg (Reference Number: PV4911) and conducted in accordance with the Declaration of Helsinki. One subject with insufficient data quality and two subjects with excessive error rates in task performance (>2 SDs over the mean in a ses-sion) were excluded. The remaining 26 subjects (18 men, range: 18–49 yrs,M= 28.5 yrs, SD= 7.9 yrs) were included in the final analysis.

Stimuli and procedure

We utilized the Bergen dichotic listening task [4,30], where six consonant-vocal (CV) syllables were coupled and presented simultaneously to each ear via closed headphones (Sennheiser, HAD 200) at 75 dB. We ruled out effects of syllable voicing by combining only syllables with

Fig 1. Dichotic listening task and procedure. (A) Exemplary single trial. After 1sec of central fixation, two syllables were presented simultaneously to both ears. After a delay, participants chose the syllable that they perceived out of six alternatives. (B) Procedure. Every subject participated in two sessions (sham and anti-phase tACS) on two different days. The order of sessions was randomized across participants. Every session started with a resting state (RS) EEG, followed by either sham or anti phase stimulation at 40Hz and another resting state EEG.

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Intrinsic phase asymmetries predict tACS effects

the same voice onset time, which yielded 12 dichotic CV pairs. Voice onset time is character-ized by the length of time between the release of a consonant and the onset of voicing. Three syllables (/ba/, /da/, /ga/) were voiced and had a short voice onset time (17-32ms), the other three syllables (/pa/, /ta/, /ka/) were unvoiced with a long voice onset time (75–80 ms). Stimu-lus onset was temporally aligned and lasted for 400-500ms.

All subjects took part in both (single-blinded) tACS sessions on two different days

(M= 2.65 days; range: 1–12 days), while the session order was counterbalanced (Fig 1B). After performing the hearing test and filling out all questionnaires, the participants performed 6 practice trials on the day of the first session to get familiarized with experimental procedure and stimulus material. 240 trials were randomly presented in 2 blocks during each tACS ses-sion (sham and anti-phase). Every trial started with the participant fixating a central fixation cross for one second, then a syllable combination was played through the headphones and par-ticipants indicated their choice from a circular formation showing all six syllables. Parpar-ticipants navigated through the alternatives by left mouse button clicks and confirmed their choice with a right button click. A fixed inter-stimulus interval of 1s was applied between the offset of the visual presentation and the ensuing auditory stimulus. Hence, the trial duration varied between 3.5 and 6.5s in dependence of the individual reaction time. After a fixed delay of 1s, the next trial started. The participants were instructed to report the syllable that they under-stood most clearly between all 6 syllables, while they were not informed that each trial con-sisted of two different syllables. Furthermore, we encouraged them to fixate on the cross, relax, reduce head and eye movement and avoid jaw muscle contraction.

We ran the experiment in an electrically shielded and soundproof cabin, where participants were seated with a distance of 60cm in front of a BenQ XL2420T screen (1920 x 1080, 120 Hz).

Stimulus presentation was controlled via Presentation software (Neurobehavioral Systems, Albany, CA).

Fig 2. Spatiotemporally-matched tACS. (A) Electrode layout. Black dots indicate the 64 EEG electrodes, while all the red dots indicate the potential positions for tACS electrode placement. (B) Targeted region of interest: area 42. (C) Result of the electric field simulation to target the left BA42. Upper: Resulting electric field on an MNI brain. Lower: 2D topography that highlights which positions should be utilized for stimulation electrode placement. Here, we constrained the electrode placement to the 4 electrodes with the highest contribution. (D) Directionality of the electric field. Note, we modeled the electric field in a way that the field lines were parallel to the assumed tangential dipole orientation in BA42. (E) Resulting asymmetric tACS electrode placement relative to other potential tACS electrode positions (black). Red and blue dots indicate opposite polarities.

https://doi.org/10.1371/journal.pone.0213996.g002

Intrinsic phase asymmetries predict tACS effects

EEG acquisition and tACS

EEG and tACS Ag/AgCl electrodes were mounted in a custom-made elastic cap for 104 elec-trodes (Easycap). EEG recordings were obtained from 64 Ag/AgCl elecelec-trodes (no amplitude clipping, impedances<15 kO, referenced to FCz) using a slightly abrasive electrolyte gel (Abralyt 2000, Easycap). EEG was recorded during all conditions (Resting State 1, Sham, Verum, Resting State 2) using BrainAmp amplifiers (Brain Products GmbH). Signals were sampled at 5 kHz, amplified in the range of±16.384 mV at a resolution of 0.5μV and stored for offline analyses.

Transcranial stimulation was applied via a battery-operated stimulator (DC-Stimulator Plus, NeuroConn) using eight Ag/AgCl electrodes (12 mm diameter, Easycap). Electrode placement was based on a current flow model, which was optimized to target the auditory cor-tex based on 40 available electrode positions (Fig 2A). The combined impedance of all elec-trodes was kept below 5 kO, as measured by the NeuroConn stimulator, using Signa

electrolyte gel (Parker Laboratories Inc.). A sinusoidally alternating current of 1,000μA (peak-to-peak) was applied at 40Hz continuously for 20 minutes during each session. During sham and real stimulation the current was ramped up over 10 seconds to 1,000μA, but discontinued during the sham condition. All subjects confirmed that stimulation was acceptable and mainly noticeable during the ramp-in phase. It did not induce painful skin sensations or phosphenes.

On debriefing, 50% of the subjects were able to correctly guess which tACS-session was assigned to T1 and T2, which confirmed that single blinding was successful.

Data analyses

The data were analysed in Matlab R2017a using the EEGlab [31] and CircStat [32] toolboxes, custom-written scripts, and the LORETA KEY software package [33] (The KEY Institute for Brain-Mind Research, Dept. of Psychiatry, University Hospital Zurich, Switzerland,http://

www.uzh.ch/keyinst/loreta.htm).

Behavioral data. We assessed the distribution between right ear and left ear reports by means of a laterality index (LI), ranging from -100 to 100 according to the following formula:

LI¼100� ðcorrect RE reports correct LE reportsÞ

ðcorrect RE reportsþcorrect LE reportsÞ ð1Þ while behavioral modulation was computed as

LI_mod¼LI_Verum LI_Sham ð2Þ

As a result, positive LI-values indicate a bias towards right ear reports; negative LI-values indi-cate more left ear reports and a value of zero signals a perfectly balanced distribution between left and right ear reports.

EEG data preprocessing. Since no artifact removal approach that reliably reconstructs EEG phase properties is known so far [34,35], we focused all EEG analyses on the sham session.

First, we removed noisy channels, downsampled the data to 250Hz and filtered the signal in the range from 1–100 Hz using two-pass finite element impulse response (FIR) filters as imple-mented in EEGLab. Moreover, we filtered out line noise at 50 Hz and its harmonics. The fil-tered data were visually inspected using the raw signal as well as a Fast Fourier transformation (FFT) to ensure that all artifacts were successfully suppressed. Then, removed channels were interpolated by spherical spline interpolation. Epochs containing saccades, noise or excessive muscle artifacts were removed after visual inspection, and all channels were re-referenced to a common average. Subsequently, an independent component analysis (ICA) was employed to

Intrinsic phase asymmetries predict tACS effects

identify blinks, eye movements, electrocardiographic and saccadic spike potential artifacts with regard to time courses, characteristic topographies and frequency distributions [36,37].

Finally, DL-data were segmented into 400ms-epochs, starting 200ms before stimulus onset (Fs = 250Hz, 100 time points), and separated by perceptual outcome (left or right ear percept).

Out of 240 trials, an average ofM±SD= 76.15±22.02 left ear trials (min: 38; max: 129) andM

±SD= 119.85±20.18 right ear trials (min: 93; max: 155) remained for the analysis of the EEG phase signature.

Importantly, the sample size bias affects the comparison of averaged electrophysiological measures in sensor space [38,39], and even more heavily in source space analyses due to its additional influence on the applied spatial filters [40]. Since matching the trial numbers across conditions within each subject would not sufficiently control for a sample size bias with respect to the ensuing circular-linear correlation analysis, we decided to rule out confounding influ-ences of unequal trial numbers on the individual phase asymmetries by randomizing across conditionsandsubjects.

Hence, we randomly subsampled 38 trials (lowest number across all subjects) out of each subject’s datapool for the left and right ear condition, respectively: In this procedure, all trials of each participant were stored in a Matlab-array, which was subsequently randomly permuted using the functionshuffle.m. The first 38 trials along each permuted trial dimension were selected for both ear conditions separately. All instances of the presented data analysis relate to the first randomly selected sample of trials. In total, an absolute number of 3120 trials was dis-carded throughout the subsampling procedure. Crucially, we repeated this subsampling proce-dure in a supplementary analysis to confirm that our results were not restricted to one trial selection (seeS2 Text,S2 FigandS2 Table).

Source space analyses. Next, the preprocessed data were projected into source space using the LORETA KEY software. We calculated a transformation matrix for all 60 electrodes using exact LORETA zero-error tomography. Based on previous findings [4,5], we decided to focus on the secondary auditory cortex (BA42) given its functional relevance in early auditory perception and syllable perception in particular [41,42]. The ROIs were defined according to the Talairach-Atlas [43] as implemented in the LORETA KEY software. Importantly, we exploited the tangential dipole activity (z-component of the current density vector) in the cen-troid voxel of BA42 because this dipole component corresponds best to the time window of interest (-200 to 200ms), hence to its underlying neural generators covering the Planum tem-porale [44–46]. Having extracted the tangential auditory dipole activity and at 40Hz, we com-puted the asymmetryΔφfor each time pointtby deriving the angleφof the complex

conjugate product of the Hilbert-transformed data with the following formula:

DφðtÞ ¼ jφðhilbertðx_leftðtÞÞ �conjugateðx_rightðtÞÞÞj ð3Þ

where

0�DφðtÞ �p ð4Þ

and

p�φðhilbertðx_leftðtÞÞ �conjugateðx_rightðtÞÞÞ �p ð5Þ

Finally, we calculated each participant’s average time course ofΔφacross trials for each time point (circ_mean.mfunction).

Statistical analyses. Unless stated otherwise, the significance level was set toα= .05 in all tests, and all mean values are reported with standard deviation values (M±SD). All circular data were processed using the CircStat toolbox. Correlations between behaviour and phase

Intrinsic phase asymmetries predict tACS effects

dynamics were assessed as:

r¼

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi r_xs² þr²_xc 2�r_xs�r_xc�r_cs

1 r_cs² s

ð6Þ

where rxc, rcsand rxsare defined as

r_xc¼corrðx;sinðφÞÞ ð7Þ

r_xs¼corrðx;cosðφÞÞ ð8Þ

r_cs ¼corrðsinðφÞ;cosðφÞÞ ð9Þ

withφbeing the circular and x being the linear variable (circ_corrcl.mfunction).

In contrast to repeated measures analyses of variance (RM-ANOVA), permutation-based cluster statistics do not depend on assumptions about the data distribution due to their non-parametric nature [47]. Thus, we assessed differences in time courses of the phase asymmetries between left and right ear trials percept trials (100 time points, -200 to 200ms) with a non-parametric permutation test for paired conditions where a permutation distribution was com-puted by randomly switching the condition labels within participants in each of 10.000 itera-tions. To address the issue of multiple comparisons, we here report the p-values using the statistics of the maximum difference (maxstat-method, see [47]) after 10.000 permutations.

Since we expected a clear right ear advantage for syllable perception in right-handed indi-viduals, we first conducted two separate t-tests (paired samples, Bonferroni-corrected) to prove that syllables were more often reported through the right ear than through the left ear during both sham- and verum-tACS.

The influence of tACS on the laterality index was assessed with a two-sided t-test for paired samples. Furthermore, the distributions of LI-values during both tACS-sessions were checked for normality with Lilliefors test. Effect sizes were quantified by means of Cohen’s d (t-test).

We additionally calculated a Bayes factor expressed asBF10for the hypothesized effect of tACS on the laterality index with a default scale factor ofr= 0.707.

Results

Behavioral performance during sham- and verum-tACS

The right ear advantage was present during both sham- (LISham:M= 23.714±18.557) and verum-tACS (LIVerum:M= 24.756±21.535) as participants perceived significantly more sylla-bles presented to the right ear (sham:M= 134±19.779; verum:M= 136±23.841) than to the left ear (sham:M= 83±22.258; verum:M= 82±24.661), which was confirmed by two-sided t-tests for both tACS-sessions (sham:t(25) = 6.480;p<.001,d= 2.43; verum:t(25) = 5.809;p<

.001d= 2.22). Moreover, behavioral performance was normally distributed during both sham-(p= .50) and verum-tACS (p= .50).

Twenty-three out of 26 participants showed a positive LI during both sessions, whereas 3 participants had a negative LI. Across all participants, reporting a syllable that was not pre-sented occurred in 9.311%±5.276% of cases during sham-tACS and in 8.862%±4.489% of cases during verum-tACS.

Intrinsic 40Hz phase asymmetries predict stimulation outcome

The main influence of tACS on behavioral performance was assessed in a two-sided t-test on the LI values during sham and verum-tACS. This did not confirm the hypothesized increase of

Intrinsic phase asymmetries predict tACS effects

the LI (Fig 3A;t(25) = 0.597,p= .556,d= 0.05), which suggests that 40Hz-tACS applied in this electrode montage (Fig 2E) did not consistently amplify the right ear advantage.

The absence of a general tACS effect on behavioral performance, as indicated by a Bayes factor ofbf10= 0.244, raised the question whether the individual stimulation outcome might depend on the inter-individual differences in oscillatory phase dynamics between the left and right secondary auditory cortices (BA42) at 40Hz. Accordingly, if interhemispheric phase dif-ferences predicted a perceptual shift to the left or the right ear, this should be indicated by a circular-linear correlation between the intrinsic phase asymmetries between the left and right BA42 at 40Hz and the difference of LI-values during verum- and sham-tACS. Hence we calcu-lated each participant’s phase asymmetry at 40Hz during the sham session by extracting the angle of the complex conjugate product of the Hilbert-transformed source space data.

After dividing all trials into left or right ear responses, circular means were calculated across trials for each time point (-200ms to 200ms post-stimulus onset interval) in each subject. We applied a non-parametric paired sample permutation test to investigate whether the across participant phase asymmetry at 40Hz differed between left and right ear percept in a specific time period. The permutation test revealed that the phase asymmetries of the perceptual out-comes differed significantly in the post-stimulus onset interval from 36-56ms (Fig 4A; LE per-cept: 79.1˚±20.8˚; RE perper-cept: 67.8˚±18.1˚; circular mean±SD; Permutation Test ’t-max’-Method, multiple comparison correctedp-values are displayed inTable 1). Clearly, the grand average phase asymmetry at 40Hz between the left and right BA42 was augmented during left ear percept compared to right ear percept in this time window. As participants with a negative LI might exhibit an atypical organization of speech perception due to an altered interhemi-spheric communication between auditory cortices [6,48], we repeated the non-parametric

Fig 3. Behavioral results. Laterality Index (LI): Positive values indicate a bias towards right ear reports. (A) 180˚ tACS at 40Hz does not increase the LI (one-sided t-test for paired-samples,t(25) = 0.597,p= .278,d= 0.05). The error bars depict the standard error of the grand average behavioral performance (LI) during both conditions (mean±SEM). (B) The individual behavioral performances (N= 26) during both conditions. The dashed lines highlight the directionality of the individual modulations (up: increase of LI; down: decrease of LI).

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Intrinsic phase asymmetries predict tACS effects

permutation test after excluding three participants with an atypical LI to rule out potential confoundations (S2 Text). Importantly, the exclusion of these participants again yielded a sig-nificant difference between the perceptual responses of the phase asymmetries in the post-stimulus onset interval from 44-60ms (seeS1 Table; Figure A inS1 Fig).

Having identified a specific time window that revealed a significant difference between con-ditions, we next tested our hypothesis that the individual auditory asymmetries predicted the behavioral modulation by tACS. Therefore, we computed one circular mean across all time points in this post-stimulus onset interval (36-56ms) across each subject’s left ear trials during

Fig 4. Oscillatory key signature of the interhemispheric phase lag. (A) Time course of the interhemispheric phase difference at 40Hz between the left and right BA42 averaged across all subjects (M±SEM) during sham-tACS. The shaded bar highlights the interval (36-56ms) where the phase shifts were statistically different between conditions (paired-sample permutation test with 10000 permutations, ’tmax’-method,^�p<.05). (B) Circular-linear correlation between the individual phase shifts during auditory processing through the left ear in the cluster-corrected time window (36-56ms) and the behavioral outcome of the 180˚

stimulation at 40Hz (ΔLI = LIVerum—LISham). The significant correlation (rho= .557,p= 0.0176) indicates that tACS amplified the right ear advantage in subjects whose oscillatory asymmetry at 40Hz was smaller (closer to 0˚) during conscious auditory processing. Contrary, augmented interhemispheric asymmetry (closer to 180˚) was associated with a shift to left ear processing. (C) tACS-effect on behavioral performance after splitting the sample at the median angle (φ= 82.11˚) into two equally sized subgroups (N= 13).

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Table 1. Correctedp-values for permutation statistics. Correctedp-values (Tmax-method) for the non-parametric paired sample permutation test (Fig 4A), which was applied to the intrinsic phase asymmetries at 40Hz during left ear and right ear processing. The permutation distribution was computed by randomly switching condition labels within participants in each of 10.000 iterations.

epoch 32-36ms 36-40ms 40-44ms 44-48ms 48-52ms 52-56ms 56-60ms

p-value .1892 .0455 .0189 .0304 .0477 .0406 .0501

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Intrinsic phase asymmetries predict tACS effects

sham-tACS and assessed the circular-linear correlation between these second order means of the intrinsic phase asymmetry and the individual tACS modulations (LImod). Interestingly, a significant correlation (rho= .557,p= 0.0176) confirmed our assumption that the behavioral modulation depended on the temporal asymmetry: Stronger phase asymmetries (closer to 180˚) were associated with a perceptual shift to left ear processing, whereas an amplification of the right ear advantage was associated with weaker phase asymmetries (closer to 0˚). This was further supported by a subgroup analysis after performing a median split on phase asymme-tries during left ear percept to divide into low (φ<82.11˚) and high (φ>82.11˚) asymmetry:

The tACS-modulation was significantly elevated in subjects with phase asymmetries above 82.11˚ (LImod:M= 4.455±8.257) compared to subjects with asymmetries below 82.11˚ (LImod: M= -2.371±8.485) (Fig 4C;t(24) = 2.079;p= .049;d= 0.815). Please note that the median split analysis was performed for illustration purpose to highlight the bidirectional impact of the stimulation. Furthermore, the significant circular-linear correlation between tACS-related behavioral modulation and the intrinsic phase asymmetry (rho= .5932,p= 0.0175) during left ear percept in the sham session was not affected by the exclusion of participants with an atypi-cal LI during the sham session (Figure B inS1 Fig).

Since the participants performed the DL task during sham- and verum-tACS on two differ-ent days, this dataset could not yield information about the test-retest reliability of the intrinsic phase asymmetry. To determine this, we analyzed pilot data (N= 18) from another experiment where the DL task was performed during 64-channel EEG recording on two different days.

Crucially, the phase asymmetry values at 40Hz exhibited a high test-retest reliability during left ear processing (rho= .8529;p= .0047; seeS3 Text,S3 Fig).

Collectively, the above findings reveal that high frequency phase asymmetries in the gamma-range exhibit different temporal profiles during ambiguous auditory perception, and that the individuality of these spectral asymmetries predicts the outcome of the electrical stim-ulation on a behavioral level.

Discussion

In this study, we tested whether (1) the transcallosal information flow between the left and right SAC can be modulated during conscious auditory perception with high-frequency tACS at 40Hz, and (2) to what extent the stimulation outcome was associated with the individual asymmetries of the spectral profiles.

Clearly, our bilateral HD-montage at a phase-lag of 180˚ failed to elicit a general effect throughout all subjects. Since the participants responded differently to our fixed stimulation protocol, we performed an exploratory source space analysis to derive an oscillatory key signa-ture of the phase asymmetry at 40Hz during dichotic listening. Our EEG-analysis of the phase dynamics demonstrated that syllable perception through the left ear does not only depend on elevated functional [4] and effective [5] gamma-band coupling, but also that its mean coupling direction at 40Hz differs significantly from right ear processing. At first sight, the finding of increased phase asymmetry during left ear percept may contradict the idea that transcranially decoupling the left and right auditory cortex with a phase-lag of 180˚ causes a shift to right ear processing. Consequently, elevated interhemispheric coupling and ensuing shift to left ear per-cept would be expected by a stimulation with zero-lag between hemispheres. In accordance with that, the original communication through coherence hypothesis (CTC,[49]) initially pro-posed zero-phase synchronization in the gamma-frequency range as the key mechanism for bidirectional coupling between two neuronal groups, whereas phase synchronization in lower frequencies was suggested for enhanced delays in increasingly distant cortico-cortical com-munication. However, more recent studies evidenced that bidirectional coupling through

Intrinsic phase asymmetries predict tACS effects

gamma-band coherence entails directedness with a systematic delay [26,50–52], and thus does not occur at zero phase. Importantly, this was further supported by EEG studies investigating dichotic listening: Increased functional connectivity during left ear percept should reflect a shift away from 0˚, because the associated metric (lagged phase synchronization,[27]) sup-presses zero phase-lag contributions [53]. In line with that, another study [5] exploiting source space effective connectivity analysis during dichotic listening revealed elevated isolated effec-tive coherence (iCoh,[28]) for left ear percept from the right to the left BA42 compared to the other direction as well as compared to perception through the right ear. Delayed (non-zero) lag inter-areal gamma-band synchronization is visible in Granger-causal influences and iCoh specifically [28], because it signifies that variance in one oscillation explains unexplained vari-ance in another oscillation several milliseconds later. Collectively, our characterization of the intrinsic phase asymmetry supports the above mentioned studies in that long-range auditory synchronization in the gamma-band range enables conscious auditory perception through the subdominant ear with a non-zero phase-lag.

Here, we characterized phase asymmetries as an oscillatory network trend which exhibited considerable inter-individual variation across our sample (range: 24˚-117˚, seeFig 4B), and argue that the assessment of phase asymmetries might be a crucial network parameter to care-fully consider, in order to optimize multi-site stimulation protocols with tailored phase-lags between the targeted oscillators. This is further supported by the fact that the asymmetry values showed a high test-retest reliability (seeS3 Text, Figure B inS3 Fig), which suggests that phase asymmetries could indeed reflect a robust auditory network trend that exhibits low intra- and high inter-individual variability in a specific frequency range.

To date, tACS is debated as a highly-promising tool to non-invasively probe the causal influence of neuronal oscillations for a variety of cognitive functions [9,54], while its impact on large-scale networks heavily depends on a broad variety of parameters such as stimulation intensity [55], waveform and envelope [56,57], network state [58,59] or the electrode montage [18,25]. So far, it appeared to be the nature of non-invasive brain stimulation that its effects on physiology and behavior are often small [60], whilst the publication bias further impedes criti-cal discussion on disadvantageous study protocols with regard to crucial stimulation parame-ters, such as intensity, montage frequency and phase-lag. In this study, our control analysis demonstrated that the behavioral outcome of the 180˚-stimulation depended on the phase asymmetry: Elevated phase asymmetry was associated with a shift to left ear processing, while the right ear advantage was amplified when the asymmetry was closer to 0˚ (Fig 4B). Conse-quently, the subgroup division at the median angle of 82.11˚ revealed a bidirectional impact of our stimulation (Fig 4C), suggesting that the asymmetric nature of conscious auditory process-ing can selectively be modulated by spatiotemporally-matched tACS. Moreover, these findprocess-ings support the concept that synchronized gamma-band activity not only mediates the integration of visual [18,25,61,62], but also auditory information from both hemispheres [63]. However, the circular-linear relationship raises the question how the external 40Hz driving force inter-acted with the intrinsic phase relationship of the neuronal oscillators in the left and right sec-ondary auditory cortex. We argue that the selective modulation of conscious auditory perception might depend on the deviation of the exogenous from the endogenous phase lag:

The interhemispheric network was prone to inhibition when the intrinsic lag differed strongly from the transcranially-induced 180˚-lag, whereas a shift to left ear percept was facilitated when the deviation of the tACS-induced lag from the intrinsic lag was low. Hence, it is con-ceivable that long-range gamma-band synchronization can be efficiently amplified if the exter-nal driving force mimics an electrical field bilaterally with the intrinsic phase asymmetry.

Accordingly, the cortical network dynamics should be most efficiently hampered if the devia-tion of the exogenous phase lag from the intrinsic lag approximatesπ.

Intrinsic phase asymmetries predict tACS effects

Since schizophrenic patients with auditory-verbal hallucinations (AVH) exhibit increased interhemispheric gamma-band coupling during dichotic listening and thus a reduced right ear advantage [64–66], the current study was initially designed to increase the laterality index, which might offer a potential application of tACS in normalizing disturbed gamma-band con-nectivity underlying AVH in patients with schizophrenia. Our results suggest that the charac-terization of the intrinsic phase relationship in the gamma-band range might benefit tailored tACS protocols in future studies.

Importantly, the interindividual variability in shape and size of the targeted pathway was highlighted by a study that utilized Diffusion Tensor Imaging (DTI) of the CC with a focus on posterior subregions connecting the auditory cortices: Stronger anatomical connectivity between these areas was associated with augmented left ear processing [67]. Even though our data do not provide tractographic information about the CC, it is conceivable that the interin-dividual differences in angular asymmetries at 40Hz might relate to ininterin-dividual variation of structural features of the transcallosal auditory pathways, and that these phase oscillations reflect undulations of neuronal excitability [68]. Such phase-related interindividual differences in the gamma-band level out in the grand average across subjects, which may explain the absence of a general behavioral effect by 180˚-tACS across all participants.

Several studies have pointed out the role of slow wave oscillatory dynamics for hearing [19,21], speech perception [24] and syllable perception in particular [69]. Here we provide evi-dence that high-frequency oscillations in the gamma-band range might not only shape audi-tory perception in terms of magnitude properties [70], but in terms of the individual

interhemispheric phase signature. In our experiment, our effects are better explained by 40Hz-phase properties given that we applied the alternating currents at equal intensities to each hemisphere, while the phase asymmetry interacted with the [53]advanced protocols can selec-tively modulate long-range cortico-cortical signal transmission with phase-dependent effects in different modalities [16,18–20,71,72].

Confounds and limitations

A number of limitations hamper the analysis of gamma-band activity and long-range coupling in human EEG recordings, such as the effects of volume conduction in the cortical tissue, broadband muscle activity that might obscure physiologic gamma-band signatures or the low spatial resolution of EEG recordings. We addressed these issues by analyzing all data at the source space level using the eLORETA approach after carefully removing artifacts by means of an ICA [37]. In addition, we employed connectivity analyses which reduce the impact of vol-ume spread and allow to estimate the directionality of these effects [53].

A further potential issue is the statistical validity of the grand average phase asymmetry time courses (Fig 4A), as each averaging and trial subsampling method has some limitations.

To control for a sample size bias, we randomly selected a subsample of 38 trials for each partic-ipant and thus discarded event-related data from ensuing analyses. Importantly, this method was exploited in another EEG study investigating long-range connectivity estimates in source space [40] to avoid an additional sample size bias to spatial filters; and was further discussed as a valid method to compute grand average images across subjects and conditions [73]. Cru-cially, matching the trial numbers within subjects would not correct for a sample size bias with respect to circular-linear correlation analyses. Furthermore, our goal was to keep results com-parable with our supplementary reliability analysis (seeS3 Text,S3 Fig), as classical test theory demands an equal number of observations throughout all subjects for the assessment of reli-ability scores [38]. In this study, we accepted a minimum number of 38 trials since previous studies had demonstrated that an adequate reliability estimate of 0.8 can be obtained at a

Intrinsic phase asymmetries predict tACS effects

Im Dokument Modulation of Interhemispheric Auditory Communication by Transcranial Alternating Current Stimulation (Seite 60-80)